There is a fundamental gap in our understanding of how HIV evades innate and adaptive immune responses to establish a persistent infection. Our laboratory's long-range goal is to provide better therapies for HIV- infected people and to move closer to a cure for this infection. We expect that a greater understanding of HIV persistence will inform the design of therapeutic strategies that will result in prevention of infection and/or long term remission or cure. As the next step towards this goal, the objective of this application is to identify cellular intrinsic antiviral mechanisms and to understand how HIV evades them. Our central hypothesis is that APOBEC 3G, an intrinsic antiviral factor that inactivates the virus by converting cytosine residues to uracils, also alerts natural killer cells to the infection. HIV counteracts this cellular response via the concerted activities of Vif, Vpr and Nef. The rationale of the proposed work is that identifying antiviral mechanisms and understanding how HIV evades them will yield mechanistic insights into the establishment of persistent infection and will hasten the discovery of disease-modifying therapies. We plan to test our central hypothesis and accomplish the objective of this application by pursuing the following three specific aims: (1) Determine whether Vif and Vpr counteract deleterious effects of uracil in HIV-infected macrophages. It has been well established that HIV-1 Vpr is necessary for efficient infection of macrophages whereas a potential role for Vpr in T lymphocytes has been ambiguous. Based on our preliminary data, we hypothesize that Vpr is needed in macrophages to counteract the deleterious effects of high concentrations of cytoplasmic dUTP found in non- dividing cells such as macrophages and/or to counteract macrophage-specific expression of APOBEC family members that are resistant to Vif. We will test this hypothesis by measuring uridine incorporation and HIV DNA stability in HIV-infected macrophages plus or minus Vpr. We will also use multiple approaches to assess the role of APOBEC family members. (2) Determine whether UNG2 is both a necessary host co-factor for Vpr-dependent repair as well as a target of Vpr-mediated degradation. Our working hypothesis, based on preliminary data, is that Vpr counteracts APOBEC-mediated cytidine deamination and incorporation of uridine by activating the uracil glycosylase, UNG2, to promote repair of viral DNA. We will use molecular approaches to determine the extent to which UNG2 is responsible for Vpr-dependent removal of uridine residues incorporated in the HIV genome in primary macrophages and T cells. The proposed experiments will help explain apparently conflicting published data regarding a role for UNG2 in HIV infection. (3) Determine whether baseline and/or induced A3G is associated with reservoir size in HIV-infected people. Our preliminary data indicate that APOBEC3G (A3G) expression is variable amongst uninfected donors and furthermore that A3G expression and NK-cell activation and lysis are induced to varying degrees upon HIV infection. Based on these preliminary data we hypothesize that a strong intrinsic antiviral immune response mediated by A3G-dependent activation of NK cells will limit reservoir size. This hypothesis will be tested using samples from HIV-infected donors analyzed ex vivo. If our hypotheses are proven correct, this work will reveal APOBEC and uracilation as key antiviral factors that promote antiviral clearance through natural killer cell activation and lysis of virally infected cells. Moreover, antiviral compounds that disrpt the activity of Nef, Vif and Vpr will limit viral infectivity and promote immune cell clearance of infected cells. These results have particular relevance for cure research as intact immune surveillance systems may be needed to identify reactivated latent reservoirs of HIV and to eradicate the infected cells.